Ubpy controls the stability of the ESCRT-0 subunit Hrs in development

Abstract

Ubiquitylated developmental membrane signaling proteins are often internalized for endocytic trafficking, through which endosomal sorting complexes required for transport (ESCRT) act sequentially to deliver internalized cargos to lysosomes. The ESCRT function in endocytic sorting is well established; however, it is not fully understood how the sorting machinery itself is regulated. Here, we show that Ubiquitin isopeptidase Y (Ubpy) plays a conserved role in vivo in the homeostasis of an essential ESCRT-0 complex component Hrs. We find that, in the absence of Drosophila Ubpy, multiple membrane proteins that are essential components of important signaling pathways accumulate in enlarged, aberrant endosomes. We further demonstrate that this phenotype results from endocytic pathway defects. We provide evidence that Ubpy interacts with and deubiquitylates Hrs. In Ubpy-null cells, Hrs becomes ubiquitylated and degraded in lysosomes, thus disrupting the integrity of ESCRT sorting machinery. Lastly, we find that signaling proteins are enriched in enlarged endosomes when Hrs activity is abolished. Together, our data support a model in which Ubpy plays a dual role in both cargo deubiquitylation and the ESCRT-0 stability during development. KEY WORDS: Developmental signaling, Endocytic machinery, ESCRT-0, Hrs, Ubpy, Drosophila INTRODUCTIONMany developmental signaling events are initiated through ligand binding to respective receptors at the plasma membrane. Following uptake into endocytic vesicles referred to as early or sorting endosomes, membrane receptors may recycle back to cell surface via tubular recycling endosomes. However, when directed by a ubiquitin (Ub) signal, receptor complexes are captured and sorted towards lysosomal degradation (Henne et al., 2011). The Ub moieties of protein cargos are recognized by ESCRT complexes, and are sorted into invaginating multivesicular bodies (MVBs). Mature MVBs fuse with lysosomes to deliver protein cargos for degradation (Raiborg and Stenmark, 2009). ESCRT-0 is composed of heterodimers of two subunits: Hrs and Stam. Both subunits bind Ub and clathrin, and Hrs additionally has an FYVE zinc-finger domain that binds phosphatidylinositol 3-phosphate. The ability to bind both lipid and Ub allows ESCRT-0 to initiate endosomal sorting (Clague et al., 2012).Ubpy (or USP8), a USP family deubiquitinase (DUB), participates in sorting of ubiquitylated receptors through its interaction with ESCRT-0 (Mizuno et al., 2005;Row et al., 2006). Most studies of Ubpy focus on endosomal trafficking of growth factor receptor tyrosine kinases (RTKs) in cultured vertebrate cells. However, conflicting data have been reported. In some cases, reduced Ubpy activity results in accumulation of ubiquitylated cargos (Bowers et al., 2006;Mizuno et al., 2006;Row et al., 2006; Alwan and van Leeuwen, 2007). Other studies suggest that Ubpy promotes RTK stability (Mizuno et al., 2005;Niendorf et al., 2007; Berlin et al., 2010). The role of Ubpy in Drosophila development is equally controversial. It was ...

The emerging role of deubiquitinating enzymes in genomic integrity, diseases, and therapeutics

“…While both K63-specific DUB AMSH and non-ub-chain specific USP8 balance the receptor degradation and recycling, exhibiting negative regulation of lysosomal sorting, the roles of AMSH and USP8 are worthy of digging at depth [29]. AMSH and USP8 showed a positive role in the downregulation of protease-activated receptor 2 and additionally, USP8 exhibits pleiotropic effects considering its regulatory role in ESCRT-0 and receptors per se [30, 31]. …”

Stuxnet Facilitates the Degradation of Polycomb Protein during Development

“…In some experiments, MG132 (50 mM; Sigma) or ALLN (50 mM; Sigma) was added to cultured cells or wing discs for 6-8 hr to inhibit proteasome activity, while E64 (50 mM; Sigma), leupeptin (50 mM; Sigma), or NH 4 Cl (10 mM) was used to disrupt lysosome function. Cycloheximide (50 mg/ml; Sigma) was added to S2 cells for 6 hr to inhibit nascent protein synthesis (Zhang et al, 2014), while colchicine (30 mM; Sigma) was added to S2 cells for 12 hr to induce mitotic arrest (Rogers et al, 2009). …”

A functional endosomal pathway is necessary for lysosome biogenesis in Drosophila

“…Earlier studies extensively showed the implication of UBPY in the endosomal pathway in both Drosophila and mammalian cultured cell models [19–26, 41, 42]. We hypothesized that the autophagy flux blockade and impaired lysosomes formation induced by Ubpy loss-of-function might be related to its function in the endosomal pathway, suggesting that the overall endosomal process is crucial for lysosomal biogenesis.…”